The ability to effectively label a target molecule with a dye is highly dependent on the reactive groups present on each of the molecules in the reaction and the conjugation conditions. Reagents such as succinimidyl esters (SE) and perfluorophenyl (PFP) esters have high reactivity rates with water, thereby limiting preparation, packaging, dispensing and purification conditions and their subsequent shelf life. In addition, due to their hydrolytic reactivity, most of the reagents used for biomolecule labeling in aqueous buffers hydrolyze prior to reaction with the desired biomolecule and are therefore wasted.
Gee et al. (Tetrahedron Letters (1999), 40, 1471-1474) describes 4-sulfotetrafluorophenyl (STP) esters for use in dye labeling. These groups have been shown to be amenable to conjugation in aqueous environments.
Koichi et al. (Chemical & Pharmaceutical Bulletin (1987), 35(3), 1044-1048) and Tsuji et al. (Peptide Chemistry (1986), Volume Date 1985, 23rd 111-114) describe peptide synthesis via ester activation with potassium dichlorophenolsulfonate, sodium dibromophenolsulfonate, and sodium nitrophenolsulfonate. No description of labeling or conjugation of molecules such as dyes is provided.
While many labeling reagents exist and have been used with intermittent success, there remains a need for labeling reagents that produce high yields under biologically relevant reaction conditions. Additionally, a need exists for a conjugation product that is stable and does not hydrolyze in aqueous environments.